The large-scale, economic purification of proteins is increasingly an important problem for the biotechnology industry. Generally, proteins are produced by cell culture, using either mammalian or bacterial cell lines engineered to produce the protein of interest by insertion of a recombinant plasmid containing the gene for that protein. Since the cell lines used are living organisms, they must be fed with a complex growth medium, containing sugars, amino acids, proteins and growth factors, usually supplied from preparations of animal serum. Concomitantly to the expression of the target protein many other proteins are also produced. Isolation of the desired protein from the mixture of compounds fed to the cells and from the by-products of the cells themselves to a level of purity sufficient for use as a human therapeutic poses a formidable challenge.
Procedures for purification of proteins from cell debris initially depend on the site of expression of the protein. Some proteins can be caused to be secreted directly from the cell into the surrounding growth media; others are made intracellularly. For the latter proteins, the first step of a purification process involves lysis of the cell, which can be done by a variety of methods, including mechanical shear, osmotic shock, or enzymatic treatments. Such disruption releases the entire protein contents of the cell into the homogenate, increasing even further the difficulty to isolate the target protein. The same problem arises, although on a smaller scale, with directly released proteins due to the natural death of cells during the culture process.
Once a clarified solution containing the protein of interest has been obtained, its separation from the other proteins produced by the cell is usually attempted using a combination and sequential application of different chromatography techniques. These techniques separate complex mixtures of proteins on the basis of their charge, degree of hydrophobicity, size, or affinity. Several different chromatography resins are available for each of these techniques, allowing accurate tailoring of the purification scheme to the particular protein involved. The essence of each of these separation methods is that proteins can be caused either to move at different rates down a long column, achieving a physical separation that increases as they pass further down the column, or to adhere selectively to the separation medium, being then differentially eluted by different solvents. In some cases, the desired protein is separated from impurities when the impurities specifically adhere to the column, and the protein of interest does not, that is, the protein of interest is present in the “flow-through.” However, often a purified protein solution containing a protein of interest still also contains a variety of contaminating proteins and other undesirable impurities—albeit at a lower amount than in the starting material for purification. Current attempts to overcome this problem typically involve a so-called polishing step. This step often involves gel filtration, for example, when the contaminating proteins have a molecular weight different from the target protein, immunoaffinity chromatography, or ion exchange chromatography.
Immunoaffinity chromatography is most useful when all contaminating proteins present in a sample are known and when antibodies against those contaminating proteins are available. Typically, antibodies are then immobilized to a solid support and used as immunosorbents. However, there are substantial drawbacks to this approach. Very often, the identity of the contaminating proteins is not known and thus, this antibody approach is not feasible. In addition, immunoaffinity columns are expensive and seldom totally specific for their target.
Anion exchange chromatography is a general approach and often used to remove endotoxins and foreign DNA, both relatively acidic molecules. This approach also binds other molecules that are acidic, such as certain proteins. However, this approach is ineffective for removal of contaminating proteins that have characteristics (e.g., same net charge) similar to a target protein of interest.
Thus, very often, contaminating proteins whose properties are not known are very difficult to remove. In the case of therapeutical protein solutions even trace amounts of contaminating proteins may have a disastrous effect on a patient to whom such therapeutical protein is administered. Such effects include severe allergic or immunological reactions. Often these effects are caused by contaminating proteins that are derived from eukaryotic or prokaryotic cells that are used to recombinantly express the therapeutical protein. These contaminating proteins are known as HCPs (Host Cell Proteins). HCPs, by definition, are very diverse and using methods of the prior art cannot be removed in a single process. Therefore their elimination is contingent upon a series of steps that also contribute to the reduction of the overall yield of the therapeutical protein of interest. Thus, methods whereby all contaminating proteins or impurities are at least partially, preferably completely, removed in a single purification step are preferred over the prior art methods.